Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue

ABSTRACT

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/211,236, filed Jul. 15, 2016, which claims priority to and thebenefit of U.S. Provisional Application No. 62/193,819, filed Jul. 17,2015; U.S. Provisional Application No. 62/261,654, filed Dec. 1, 2015;U.S. Provisional Application No. 62/277,283, filed Jan. 11, 2016; andU.S. Provisional Application No. 62/323,018, filed Apr. 15, 2016. Eachof the above-mentioned applications is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

Standard immunohistochemical methods allow for simultaneous detectionof, at most, six to ten protein targets, with three to four targetsbeing typical. There exists a need for probes, compositions, methods,and kits for simultaneous, multiplexed detection and quantification ofprotein expression in a user-defined region of a tissue, user-definedcell, and/or user-defined subcellular structure within a cell.

SUMMARY OF THE INVENTION

The present invention relates to probes, compositions, methods, and kitsfor simultaneous, multiplexed detection and quantification of proteinexpression in a user-defined region of a tissue, user-defined cell,and/or user-defined subcellular structure within a cell.

An aspect of the present invention relates to a method including stepsof (1) contacting at least one protein target in or from at least onecell in a tissue sample with at least one probe comprising atarget-binding domain and a signal oligonucleotide; (2) providing aforce to a location of the tissue sample sufficient to release thesignal oligonucleotide; and (3) collecting and identifying the releasedsignal oligonucleotide, thereby detecting the at least one proteintarget in or from a specific location of the tissue sample that wasprovided the force. The specific location is a user-defined region of atissue, user-defined cell, and/or user-defined subcellular structurewithin a cell. The target-binding domain comprises a protein-bindingmolecule, e.g., an antibody, a peptide, an aptamer, and a peptoid. Inembodiments, two or more protein targets are detected. In embodiments,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ormore targets, and any number therebetween, are detected; for example,800 or more different targets can be detected. In embodiments, detectingincludes quantifying the abundance of each target.

In embodiments, the method further includes repeating at least steps (2)and (3) on at least a second specific location of the tissue sample, thesecond specific location comprising at least a second cell. Inembodiments, detecting includes comparing the abundance of the at leastone protein target in or from the first specific location and in or fromthe at least second specific location. The at least one cell and atleast second cell may be the same cell type or distinct cell types. Insome embodiments, detecting includes quantifying the abundance of the atleast one protein target in or from a first cell type and in or from theat least a second cell type. In embodiments, first and second cell typesare independently selected from a normal cell and an abnormal cell,e.g., a diseased and cancerous cell.

In embodiments, the at least one cell is directly immobilized to asurface or is indirectly immobilized to the surface via at least oneother cell. A tissue sample may be a 2 to 1000 μm thick tissue section,e.g., obtained from a formalin-fixed paraffin embedded (FFPE) sample orfrom an unfixed sample. The at least one cell may be fixed or unfixed.The at least one cell may be stained or labeled prior to step (2)allowing visualization of a subcellular or cellular structure in thestained or labeled cell. Alternately, for tissue sections, a sectionadjacent to the section that is contacted with the probes may be stainedor labeled prior to step (2), thereby allowing estimation of asubcellular, cellular, or tissue-related structure in the correspondingcell or nearby cell in the section that is contacted with the probes.Such staining or labeling techniques are well known in the art.

In the above aspect, at least one probe further includes a linker (e.g.,a cleavable linker) located between the target-binding domain and thesignal oligonucleotide. The cleavable linker may be photo-cleavable,which is cleaved by light provided by a suitable coherent light source(e.g., a laser and a UV light source) or a suitable incoherent lightsource (e.g., an arc-lamp and a light-emitting diode (LED)). The lightsource may irradiate at least one subcellular structure of the at leastone cell and the abundance of the at least one protein target in or fromthe at least one subcellular structure of the at least one cell can bedetected. Also, the light source may first irradiate at least onesubcellular structure in the at least one cell and later irradiate atleast one subcellular structure in the at least second cell, allowing acomparison of the abundance of the at least one protein target in orfrom the at least one subcellular structure in the at least one cell andthe at least one subcellular structure in the at least second cell.

In embodiments, the signal oligonucleotide is a single-stranded nucleicacid or a partially double-stranded nucleic acid.

In embodiments, the sample may be cultured cells or dissociated cells(fixed or unfixed) that have been immobilized onto a slide. The samplemay comprise cells (including both primary cells and cultured celllines) and/or tissues (including cultured or explanted). The sample maycomprise a cultured cell, a primary cell, or a dissociated cell from anexplant.

In embodiments, the illumination of a region of interest smaller that afield of view (for example a single cell or a subcellular structurewithin a cell) comprises use of a laser scanning device (e.g., confocal)or a digital mirror device (DMD) to direct the light.

In embodiments, a probe is prepared by a cysteine bioconjugation methodthat is stable, site-specific to, preferably, the antibody'shinge-region heavy-chain. In embodiments, a probe can comprise aplurality (i.e., more than one, e.g., 2, 3, 4, 5, or more) labeledoligonucleotides per antibody.

Detecting comprises a polymerase reaction, a reverse transcriptasereaction, hybridization to an oligonucleotide microarray, massspectrometry, hybridization to a fluorescent molecular beacon, asequencing reaction, or nCounter® Molecular Barcodes. In preferredembodiments, nCounter® systems and methods from NanoString Technologies®are used.

In embodiments, the signal oligonucleotide is collected from a tissuevia liquid laminar, turbulent, or transitional flow. The flow may be viaa channel, e.g., having 25 to 500 μm depth between the tissue and afluidic device or impermeable barrier placed over the tissue.

In embodiments, the signal oligonucleotide is collected from a solutionproximal to, e.g., at least immediately above, the at least one cell.The proximal solution may be collected by aspirating, e.g., via apipette, a capillary tube, a microarray pin, a flow cell comprisingholes, or another suitable aspirating system known in the art or anycombination thereof. The capillary tube may comprise an optical devicecapable of transmitting a light force, e.g., UV light, to the at leastone cell. The pipette or a microarray pin may be attached to an arraycomprising a plurality of pipettes or microarray pins. The proximalsolution may comprise an anionic polymer, e.g., dextran sulfate, and/orsalmon sperm DNA and/or the collected signal oligonucleotide may beadded to a solution comprising an anionic polymer, e.g., dextransulfate, and/or salmon sperm DNA. Other non-specific blocking agentsknown in the art in addition to or instead of salmon sperm DNA may beused.

In embodiments, the method provides simultaneous spatially-resolvedprotein detection of a tissue sample.

In embodiments, digital readout comprises a linear dynamic range ofgreater than or equal to 5 logs.

In embodiments, probes are provided to a sample at concentrationstypically less than that used for immunohistochemistry (IHC) or for insitu hybridization (ISH). Alternately, the concentration may besignificantly less than that used for IHC or ISH. For example, the probeconcentration may be 2 fold less, 5 fold less, 10 fold less, 20 foldless, 25 fold less, 30 fold less, 50 fold less, 60 fold less, 70 foldless, 80 fold less, 90 fold less, 100 fold less, 200 fold less, 300 foldless, 400 fold less, 500 fold less, 600 fold less, 700 fold less, 800fold less, 900 fold less, 1000 fold less, 2000 fold less, or less andany number in between. In embodiments, probes are provided at aconcentration of 100 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM,0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM,and less and any concentration in between.

In embodiments, a tissue sample is attached to a slide and is firstimaged using fluorescence (e.g., fluorescently-labeled antibodies andfluorescent stains (e.g., DAPI)) and then expression of proteins isdigitally counted from the sample.

In embodiments, a negative purification, e.g., comprising an affinitypurification method comprising contacting intact probe molecules with animmobilized oligonucleotide that is complementary to a portion of theintact probe or an immobilized antibody or protein-binding motif thatrecognizes and binds to a portion of the intact probe, is used to removeintact probe molecules from the released signal oligonucleotides. Inembodiments, the intact probe's target binding domain comprises auniversal purification tag or sequence that is partially complementaryto the immobilized oligonucleotide or is capable of being recognized orbound by the immobilized antibody or protein-binding motif. Any such tagor sequence well-known in the art may be used in these embodiments.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein. While the disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the disclosure, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows two exemplary probes. Nucleic acid backbones (eithersingle-stranded DNA or single-stranded RNA) are shown as a straight,black line. The probes each include a target-binding domain, shown inred. The top probe includes labeled RNA segments hybridized to thenucleic acid backbone whereas the bottom probe includes labeled DNAoligonucleotides hybridized to the nucleic acid backbone. A cleavablemotif (e.g., a cleavable linker, not shown) may be located between thebackbone and a target-binding domain or within the backbone. Thecleavable motif allows release of a signal oligonucleotide from a boundtarget nucleic acid or protein; then, the signal oligonucleotide iscollected and detected.

FIG. 2 shows a first type of a probe that can bind directly to a targetnucleic acid (top). In the below image, the probe has bound to thetarget nucleic acid, shown as a blue curvilinear line. In this figureand in later figures, a reporter probe includes six positions hybridizedto labeled oligonucleotides (identified by a colored circle). Since theprobe comprises positions that can be hybridized to labeledoligonucleotides, the probe can also be referred to as a reporter probe.

FIG. 3 shows a first type of dual-probe composition of the presentinvention. Here, the first type of probe binds directly to a targetnucleic acid and a first type capture probe binds directly to the targetnucleic acid. The capture probe may include at least one affinityreagent, which is shown as an asterisk. The target nucleic acid in asample is shown as a blue curvilinear line.

FIG. 4 shows a second type of a probe (or reporter probe) that can bindindirectly to a target nucleic acid in a sample (top). Here, the probe'starget is an intermediary oligonucleotide, shown in green, which in turnbinds to the target nucleic acid in a sample, shown as a bluecurvilinear line in the bottom image. It could be said that theintermediary oligonucleotide is a probe, as defined herein, since itcomprises a nucleic acid backbone and is capable of binding a targetnucleic acid.

FIG. 5 shows a second type of dual-probe composition of the presentinvention. Here, the second type of probe binds indirectly to a targetnucleic acid in a sample (via an intermediary oligonucleotide, shown ingreen) and a second type capture probe binds indirectly to the targetnucleic acid in the sample (via another intermediary oligonucleotide,shown in orange). The capture probe may include at least one affinityreagent, which is shown as an asterisk.

FIG. 6 shows release of signal oligonucleotides from second type probes(illustrated in FIG. 4) that are bound indirectly to a target nucleicacid in a sample. The location of a cleavable motif within a probe (orin a reporter probe) affects which material is included with a releasedsignal oligonucleotide.

FIG. 7 shows three types of probes used for detecting proteins. In thetop configuration, a probe comprises a nucleic acid attached to aprotein-binding domain; in this configuration, a cleavable motif (e.g.,a cleavable linker, not shown) may be included between the nucleic acidand protein-binding domain or within the nucleic acid itself. In themiddle configuration, a protein-binding domain is attached to a nucleicacid and a probe hybridizes to the nucleic acid. The probe (comprisingthe target-binding domain and the nucleic acid attached to theprotein-binding domain (shown in green)) can be bound by a probe beforeor after the target binding domain binds a protein target (As shown inFIG. 8). A cleavable motif may be included in either or both of thebackbone or the nucleic acid attached to the protein-binding domain. Thefirst or second type probe shown in FIGS. 2 and 4 may be used in thisconfiguration for detecting a protein. In the bottom configuration, aprotein-binding domain is attached to a nucleic acid and an intermediaryoligonucleotide (shown in red) hybridizes to both a probe and to thenucleic acid attached to the protein-binding domain. The first or secondtype probe shown in FIGS. 2 and 4 may be used in this configuration fordetecting a protein.

FIG. 8 shows the middle and bottom probes of FIG. 7. The top two imagesshow the probe before and after it has bound a protein. The next imageshows the probe after its cleavable motif has been cleaved; in thisimage the cleavable motif is between the nucleic acid and the targetbinding domain. Once the nucleic acid has been released, it can beconsidered a signal oligonucleotide. In the bottom image, the signaloligonucleotide (released nucleic acid of the probe) is bound by areporter probe (e.g., as shown in FIGS. 2 and 4).

FIG. 9 shows release of signal oligonucleotides from a probe of themiddle configuration shown in FIG. 7 and the probes of FIG. 8. Thelocation of a cleavable motif within a probe (or in a reporter probe)affects which material is included with a released signaloligonucleotide.

FIG. 10 shows steps in a method of the present invention in which signaloligonucleotides from one region-of-interest (ROI) are detected.

FIG. 11 shows steps in a method of the present invention in whichregions-of-interest are located on a first serial section of a tissuesample and probes are applied to a second serial section of the tissuesample. Signal oligonucleotides are released and collected from probesbound to targets in a first region-of-interest of the second serialsection. Then, signal oligonucleotides are released and collected fromprobes bound to targets in a second (up to the n^(th))region-of-interest of the second serial section.

FIG. 12 shows multiplexed detection of a plurality of target nucleicacids and/or proteins from a first region-of-interest followed bymultiplexed detection of the plurality of target nucleic acids and/orproteins from a second region-of-interest.

FIG. 13 illustrates steps in methods of the present invention. Themethod shown may be referred herein as “nCounter® Digital MultiplexedImmunohistochemistry (IHC)”.

FIG. 14 is a flow chart demonstrating the simplified workflow and highermultiplexing capable with nCounter® Digital Multiplexed IHC (top) whencompared to standard TSA-based multiplexed IHC (bottom).

FIG. 15 are photographs showing a digital mirror device (DMD) attachedto a Ti-E microscope (top) and a brightfield image of a FFPE tissuesection (bottom). Light illumination (white spots) on the FFPE tissue(bright field image) shows multiple ROIs of about ˜10-20 μm in size,i.e., the size of a single cell.

FIG. 16 illustrates components and light paths involved with the presentinvention when the method includes use of a digital mirror device (DMD).Wide-field illumination with the DMD focused onto sample. LED providessufficient illumination to excite whole field of view at once and withsingle cell illumination such that ˜80-600 DMD pixels illuminate a 10 μmdiameter cell. A normal-grade DMD will provide sufficient single-cellresolution. DS: Dichroic mirror, FW: Filter wheel, and DMD: Digitalmirror device.

FIG. 17 illustrates components and light paths involved with the presentinvention when the method includes use of a laser scanning device (e.g.,confocal scanning device). In a confocal scanning configuration,galvo-mirrors direct light. This method requires an inexpensive 405 nmlaser. DS: Dichroic mirror, FW: Filter wheel, and MM: Motorized mirror.

FIG. 18 shows a photomicrograph establishing overall tissue morphologyof a tonsil sample that was initially imaged using two-colorfluorescence of Ki-67 (cell proliferation marker; in green) and CD3(immune cell marker; in red). Twelve regions (including the four regionsmagnified in FIG. 19) are identified with white boxes.

FIG. 19 is a graph showing nCounter® data counts of Ki-67 and CD3 forfour regions shown in FIG. 18. Images were obtained from serial sections(to allow various additional controls to be examined). In general,samples can be imaged with fluorescent antibodies and then digitallycounted (via uv-exposure) using the same slide. Multiple targetsanalyzed across twelve regions (including the four regions shown here)show distinct profiles of localization of Ki-67 and CD3. Below the graphare magnifications of the four regions.

FIG. 20 shows exemplary counts from a 30-plex oligo-antibody cocktail onthe twelve regions of interest (ROI) from the tonsil sample shown inFIG. 18. Data was obtained from serial sections (to allow variousadditional controls to be examined).

FIG. 21 shows a photomicrograph establishing overall tissue morphologyof T cells in a melanoma sample from a lymph node that was initiallyimaged using three-color fluorescence of CD3 (in red), CD8 (in green),and DAPI (in blue). The white circle is 25 μm in diameter and surroundsthree cells.

FIG. 22 shows nCounter® data of CD3 conjugate release from FFPE lymphnode tissue sections (5 μm thickness) as a function of UV illuminationarea (100 μm to 1 mm in diameter). The field diaphragm size is shownbelow the figure.

FIG. 23 shows nCounter® data for CD45 conjugate release from FFPE lymphnode tissue sections (5 μm thickness) as a function of UV illuminationarea (100 μm to 1 mm in diameter) and from the same experiment as shownin FIGS. 21 and 22.

FIG. 24 shows nCounter® data for PD1 conjugate release from FFPE lymphnode tissue sections (5 μm thickness) as a function of UV illuminationarea (100 μm to 1 mm in diameter) and from the same experiment as shownin FIGS. 21 to 23.

FIG. 25 shows a tissue microarray (TMA; left panel) of breast tumortissue containing variable levels of Her2 protein as shown in thephotomicrograph (center panel) which identifies Her2 fluorescence by IHCstaining. The right panel shows a magnification of a single region ofthe central panel.

FIG. 26 shows nCounter® count data for forty-eight representativeregions versus Her2 status (ASCO-CAP guidelines).

FIG. 27 shows Plots of nCounter® Counts versus Sum Pixel Intensities(x10³) for the forty-eight regions mentioned with respect to FIG. 26.

FIG. 28 is a photomicrograph establishing overall tissue morphology of amelanoma sample that was initially imaged using two-color fluorescenceof CD3 (in red) and DAPI (in blue). Ten exemplary regions are identifiedwith white boxes.

FIG. 29 shows exemplary counts from a 30-plex oligo-antibody cocktail onthe ten regions of interest (ROI) from the sample shown in FIG. 28.

FIG. 30A and FIG. 30B are photomicrographs showing UV illumination usinga digital mirror device (DMD) of single cells (in blue) in a tonsiltissue sample (in green).

FIG. 31A and FIG. 31D are photomicrographs showing UV illumination usinga digital mirror device (DMD) of single cells (in bright white) in atonsil tissue sample. FIG. 31B highlights the single cells noted in FIG.31A. FIG. 31D highlights the single cell noted in FIG. 31C.

FIG. 32 shows steps in a spatially-resolved FFPE Tissue Protein Assay.The steps are similar to those of a nucleic acid-detecting assay except,in the nucleic acid-detecting assay, the sample is bound with a probecomprising a nucleic acid target-binding domain rather than an antibody.

FIG. 33 shows steps in a spatially-resolved FFPE Tissue Protein Assay.

FIG. 34 shows data from an embodiment in which a whole tissue or wholesample is illuminated, e.g., with a standard UV gel box, to releasesignal oligonucleotides previously attached to a probe.

FIG. 35 shows an embodiment in which a portion of a tissue or sample isilluminated, e.g., with a microscope, i.e., UV cleavage under Microscope(Time titration experiment).

FIG. 36 shows an embodiment in which a portion of a tissue or sample isilluminated, e.g., with a microscope, i.e., UV cleavage under Microscope(Illumination area titration experiment).

FIG. 37 shows an embodiment in which a portion of a tissue or sample isilluminated, e.g., with a microscope, i.e., UV cleavage under microscope(Illumination area titration experiment—multiple targets).

FIG. 38 shows an embodiment in which a region of interest in a tissue(e.g., a breast cancer sample) is first identified for expression of amarker and this region of interest is then illuminated (e.g., with UV)to release signal oligonucleotides from a probe.

FIG. 39 shows an embodiment in which a tissue is embedded in flow cell.Data for multiple fractions is shown. As with the data of FIG. 38, herea region of interest is pre-identified for expression of afluorescently-labeled marker. Also shown are photographs and a schematicshowing configuration of the apparatus.

FIG. 40 shows an embodiment in which a tissue is embedded in flow cellwith small holes. Also shown are photographs and a schematic showingconfiguration of the apparatus.

FIG. 41A shows photographs and a schematic showing the configuration ofthe apparatus in embodiments using a flow cell with small holes. FIG.41B and FIG. 41C are plots of data collected using embodiments using aflow cell with small holes showing significant signal to noiseimprovement compared to collection of eluate from entire surface oftissue.

FIG. 42A shows data in the embodiments using a flow cell with smallholes (12 hole format) FIG. 42B shows data in the embodiments using aflow cell with small holes (96 hole format). FIG. 42C shows data in theembodiments using a flow cell with small holes.

FIG. 43A shows data in comparing background signal from flow cells inwhich whole tissue elution was performed. FIG. 43B shows data comparingbackground signal from flow cells in which elution occurred directlyabove a region of interest.

FIG. 44 is a schematic showing eluent collection with an open surfacefor a multi-region of interest aspiration embodiment. Here is shown amulti-tube array for aspiration/dispensing eluents with rotary valveselection.

FIG. 45 includes photographs and a schematic showing an embodiment inwhich eluent collection is through a capillary (micro-aspirator).

FIGS. 46A and FIG. 46B show data from the embodiment of FIG. 45 in whicheluent collection is through a capillary (micro-aspirator).

FIG. 47 is a schematic showing eluent collection with an open surfacefor a multi-region of interest aspiration embodiment or for a singleregion of interest. Here is shown a multi-tube array using pipetting vscapillary action for aspiration/dispensing and a single tube/pipet withfixed position.

FIG. 48 is a schematic showing illumination and fluid collection througha combined capillary and lens.

FIG. 49 is a schematic showing steps in an embodiment of aspatially-resolved FFPE tissue assay comprising a 96 well grid.

FIG. 50 shows protein expression data obtained from a single cell or twocells using the herein described methods and apparatuses.

FIG. 51 identifies regions of interests located on serial sections froma single tumor sample.

FIG. 52 shows counts obtained for six of the nine RNA probes included inthe assay of Example 16.

FIG. 53 shows the averages and standard deviations of counts shown inFIG. 52.

FIG. 54 shows RNA expression data and protein data for probes that weresimultaneously hybridized to nCounter® Molecular Barcodes, and digitallycounted by an nCounter® system from NanoString Technologies®.

FIG. 55 shows RNA expression data obtained from single-stranded DNAprobes and partially double-stranded DNA probes.

FIG. 56 shows RNA expression data obtained from probes hybridized in thepresent of salmon sperm DNA.

FIG. 57 shows RNA expression data from a probe specific to PSA(Prostate-Specific Antigen).

FIG. 58 shows specificity of probes increase at non-standard, sub-nMconcentrations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on probes, compositions, methods,and kits for simultaneous, multiplexed detection and quantification ofprotein and/or nucleic acid expression in a user-defined region of atissue, user-defined cell, and/or user-defined subcellular structurewithin a cell.

The present invention provides a comparison of the identity andabundance of target proteins and/or target nucleic acids present in afirst region of interest (e.g., tissue type, a cell (including normaland abnormal cells), and a subcellular structure within a cell) and theidentity and abundance of target proteins and/or target nucleic acidspresent in a second region of interest. There is no pre-defined upperlimit to the number of regions of interest and comparisons that can bemade; the upper limit relates to the size of the region of interestrelative the size of the sample. As examples, when a single cellrepresent a region of interest, then a section may have hundreds tothousands of regions of interest; however, if a tissue section includesonly two cell types, then the section may have only two regions ofinterest (each including only one cell type).

The present invention provides a higher degree of multiplexing than ispossible with standard immunohistochemical or in situ hybridizationmethods. Standard immunohistochemical methods allow for maximalsimultaneous detection of six to ten protein targets, with three to fourprotein targets being more typical. Similarly, in situ hybridizationmethods are limited to simultaneous detection of fewer than ten nucleicacid targets. The present invention provides detection of largecombinations of nucleic acid targets and/or protein targets from adefined region of a sample. The present invention provides an increasein objective measurements by digital quantification and increasedreliability and consistency, thereby enabling comparison of resultsamong multiple centers.

The probes of the present invention may have nucleic acid backbones(single-stranded DNA or RNA) having defined positions capable of beinghybridized (non-covalently bound) with at least one labeledoligonucleotide. See, FIG. 1. Such probes (which have defined positionscapable of being hybridized with at least one labeled oligonucleotideare also referred herein as reporter probes. The number of positions ona reporter probe's backbone ranges from 1 to 100 or more. Inembodiments, the number of positions ranges from 1, 2, 3, 4, 5, 6, 7, 8,9, 10 to 15, 20, 30, 40, or 50, or any range in between. Indeed, thenumber of positions (for detecting a target nucleic acid and/or fordetecting a target protein) on a backbone is without limit sinceengineering such a backbone is well-within the ability of a skilledartisan. The number of target nucleic acids and/or proteins detectableby a set of probes depends on the number of positions included in theprobes' backbones.

As used herein a labeled oligonucleotide relates to an RNA segmentincluding a detectable label or a DNA oligonucleotide including adetectable label.

A position of a nucleic acid backbone may be hybridized (non-covalentlybound) with at least one labeled oligonucleotide. Alternately, aposition may be hybridized with at least one oligonucleotide lacking adetectable label. Each position can hybridize to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 to 100 labeled (orunlabeled) oligonucleotides or more. The number of labeledoligonucleotides hybridized to each position depends on the length ofthe position and the size of the oligonucleotides. A position may bebetween about 300 to about 1500 nucleotides in length. The lengths ofthe labeled (or unlabeled) oligonucleotides vary from about 20 to about1500 nucleotides in length. In embodiments, the lengths of labeled (orunlabeled) oligonucleotides vary from about 800 to about 1300ribonucleotides. In other embodiments, the lengths of labeled (orunlabeled) oligonucleotides vary from about 20 to about 55deoxyribonucleotides; such oligonucleotides are designed to havemelting/hybridization temperatures of between about 65 and about 85° C.,e.g., about 80° C. For example, a position of about 1100 nucleotides inlength may hybridize to between about 25 and about 45 oligonucleotidescomprising, each oligonucleotide about 45 to about 25deoxyribonucleotides in length. In embodiments, each position ishybridized to about 34 labeled oligonucleotides of about 33deoxyribonucleotides in length. The labeled oligonucleotides arepreferably single-stranded DNA.

Each labeled oligonucleotide may be labeled with one or more detectablelabel monomers. The label may be at a terminus of an oligonucleotide, ata point within an oligonucleotide, or a combination thereof.Oligonucleotides may comprise nucleotides with amine-modifications,which allow coupling of a detectable label to the nucleotide.

Labeled oligonucleotides of the present invention can be labeled withany of a variety of label monomers, such as a fluorochrome, quantum dot,dye, enzyme, nanoparticle, chemiluminescent marker, biotin, or othermonomer known in the art that can be detected directly (e.g., by lightemission) or indirectly (e.g., by binding of a fluorescently-labeledantibody). Preferred examples of a label that can be utilized by theinvention are fluorophores. Several fluorophores can be used as labelmonomers for labeling nucleotides including, but not limited to,GFP-related proteins, cyanine dyes, fluorescein, rhodamine, ALEXAFlour™, Texas Red, FAM, JOE, TAN/IRA, and ROX. Several differentfluorophores are known, and more continue to be produced, that span theentire spectrum.

Labels associated with each position (via hybridization of a positionwith a labeled oligonucleotide) are spatially-separable andspectrally-resolvable from the labels of a preceding position or asubsequent position. An ordered series of spatially-separable andspectrally-resolvable labels of a probe is herein referred to as barcodeor as a label code. The barcode or label code allows identification of atarget nucleic acid or target protein that has been bound by aparticular probe.

The labeled oligonucleotides hybridize to their positions under astandard hybridization reaction, e.g., 65° C., 5xSSPE; this allows forself-assembling reporter probes or probes. Probes using longer RNAmolecules as labeled oligonucleotide (e.g., as described inUS2003/0013091) must be pre-assembled at a manufacturing site ratherthan by an end user and at higher temperatures to avoid cross-linking ofmultiple backbones via the longer RNA molecules; the pre-assembly stepsare followed by purification to remove excess un-hybridized RNAmolecules, which increase background. Use of the short single-strandedlabeled oligonucleotide (e.g., comprising deoxyribonucleotides) greatlysimplifies the manufacturing of the probes and reduces the costsassociated with their manufacture.

In embodiments, probes are provided to a sample at concentrationstypically less than that used for immunohistochemistry (IHC) or for insitu hybridization (ISH). Alternately, the concentration may besignificantly less than that used for IHC or ISH. For example, the probeconcentration may be 2 fold less, 5 fold less, 10 fold less, 20 foldless, 25 fold less, 30 fold less, 50 fold less, 60 fold less, 70 foldless, 80 fold less, 90 fold less, 100 fold less, 200 fold less, 300 foldless, 400 fold less, 500 fold less, 600 fold less, 700 fold less, 800fold less, 900 fold less, 1000 fold less, 2000 fold less, or less andany number in between. In embodiments, probes are provided at aconcentration of 100 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM,0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM,and less and any concentration in between.

Probes can be detected and quantified using commercially-availablecartridges, software, systems, e.g., the nCounter® System using thenCounter® Cartridge.

Background noise, during protein detection, can be reduced by performinga negative purification of the intact probe molecule. This can be doneby conducting an affinity purification of the antibody orphoto-cleavable linker after collection of eluate from a region ofinterest. Normally, released signal oligonucleotides will not be pulledout of solution. A protein-G or -O mechanism in a pipet tip, tube, orplate can be employed for this step. Such devices and reagentscommercially available.

Background noise, during nucleic acid detection, can be reduced byperforming a negative purification of the intact probe molecule. Thiscan be done by conducting an affinity purification of the target bindingdomain or photo-cleavable linker after collection of eluate from aregion of interest. Normally, released signal oligonucleotides will notbe pulled out of solution. To assist in the negative purification, auniversal purification sequence may included in a probe, e.g., in thetarget binding domain.

FIG. 1 shows two exemplary probes including a single-stranded nucleicacid backbone and a target-binding domain, shown in red. The top probeincludes labeled RNA segments hybridized to positions in the backbonewhereas the bottom probe includes labeled DNA oligonucleotideshybridized to positions in the nucleic acid backbone. The colors shownin FIG. 1, and elsewhere in this disclosure, are non-limiting; othercolored labels and other detectable labels known in the art can be usedin the probes of the present invention.

Probes of the present invention can be used for detecting a targetnucleic acid. FIGS. 2 and 4 illustrate this aspect. Such a probeincludes at least a backbone and a target nucleic acid-binding region.The target nucleic acid-binding region is preferably at least 15nucleotides in length, and more preferably is at least 20 nucleotides inlength. In specific embodiments, the target nucleic acid-binding regionis approximately 10 to 500, 20 to 400, 25, 30 to 300, 35, 40 to 200, or50 to 100 nucleotides in length. Probes and methods for binding andidentifying a target nucleic acid have been described in, e.g.,US2003/0013091, US2007/0166708, US2010/0015607, US2010/0261026,US2010/0262374, US2010/0112710, US2010/0047924, and US2014/0371088, eachof which is incorporated herein by reference in its entirety.

A protein target may be an intact protein, a plurality of polypeptides,a polypeptide, or a peptide.

The probes of the present invention can be used to directly hybridize toa target nucleic acid. FIG. 2 illustrates a probe (or composition) ofthis embodiment. The probes include a target nucleic-acid bindingdomain, shown in red. The target nucleic acid is shown as a bluecurvilinear line. FIG. 3 illustrates a dual probe composition includingthe probe of FIG. 2 and a capture probe. The capture probe comprises atleast one affinity reagent, shown as an asterisk. The at least oneaffinity moiety may be attached to the capture probe by covalent ornon-covalent means. Various affinity moieties appropriate forpurification and/or for immobilization are known in the art. Preferably,the affinity moiety is biotin, avidin, or streptavidin. Other affinitytags are recognized by specific binding partners and thus facilitateisolation and immobilization by affinity binding to the binding partner,which can be immobilized onto a solid support. In these figures, eachprobe includes six positions hybridized to labeled oligonucleotides,each positions is identified by a colored circle.

Any probe of the present invention may comprise an affinity moiety.

The probes of the present invention can be used to indirectly hybridizeto a target nucleic acid present in a sample (via an intermediaryoligonucleotide). FIG. 4 illustrates a probe (or composition) of thisembodiment. The probes include a target nucleic-acid binding domain,shown in red, which binds to a synthetic oligonucleotide (theintermediary oligonucleotide; shown in green) that in turn binds to atarget nucleic acid in a biological sample. It could be said that theintermediary oligonucleotide is a probe, as defined herein, since itcomprises a nucleic acid backbone and is capable of binding a targetnucleic acid. The target nucleic acid present in a biological sample isshown as a blue curvilinear line. FIG. 5 illustrates a dual-probecomposition including the probe of FIG. 4 and a capture probe. In theseembodiments, a probe's target nucleic acid-binding region hybridizes toa region of an intermediary oligonucleotide (i.e., a syntheticoligonucleotide) which is different from the target nucleic acid presentin a sample. Thus, the probe's target binding region is independent ofthe ultimate target nucleic acid in the sample. This allows economicaland rapid flexibility in an assay design, as the target (present in asample)-specific components of the assay are included in inexpensive andwidely-available synthetic DNA oligonucleotides rather than the moreexpensive probes. Such synthetic oligonucleotides are simply designed byincluding a region that hybridizes to the target nucleic acid present ina sample and a region that hybridizes to a probe. Therefore, a singleset of indirectly-binding probes can be used to detect an infinitevariety of target nucleic acids (present in a sample) in differentexperiments simply by replacing the target-specific (synthetic)oligonucleotide portion of the assay.

A probe or probe of the present invention can include a region whichpermits the release of a signal oligonucleotide following theapplication of a suitable force. In one non-limited example, the regionis a cleavable motif (e.g., a restriction enzyme site or cleavablelinker). The cleavable motif allows release of a signal oligonucleotidefrom a bound target nucleic acid or protein and the signaloligonucleotide is then collected and detected. As used herein a signaloligonucleotide is a region of a probe that presently has positionshybridized with at least one labeled oligonucleotide or is a region of aprobe (e.g., a nucleic acid molecule) that can be released from thetarget-binding domain of the probe. A signal oligonucleotide is said tobe releasable when it can be separated (i.e., cleaved and released) fromthe remainder of the probe. Examples of cleavable motives include butare not limited to photo-cleavable linkers.

In a probe of the present invention (as described herein), the cleavablemotif may be located between a nucleic acid and a target binding domain,the backbone and a target-binding domain, or within the backbone. InFIG. 6, non-limiting options for a cleavable motif's position can beinferred from gaps within a probe or a gap within an intermediaryoligonucleotide.

Probes of the present invention can be used for detecting a targetprotein. FIG. 7 illustrates probes (or compositions) of this embodiment.Such probes include at least a backbone and a target protein-bindingregion. In protein-targeting probes of the present invention, a signaloligonucleotide may the nucleic acid attached to the protein-bindingdomain. In these probes, the signal oligonucleotide is targeted andbound by a probe that comprises positions for hybridizing to labeledoligonucleotides. Such a probe is shown in FIG. 7, middle image. There,the signal oligonucleotide is seen as a green line. The probe may bebound by a probe before the probe (via its protein-binding domain) bindsa protein or afterward it binds the protein. The signal oligonucleotideneed not be bound by the probe until it has already been released fromthe target-binding domain (this embodiment is not shown).

A probe's region capable of binding to a target protein includemolecules or assemblies that are designed to bind with at least oneprotein target protein, at least one protein target protein surrogate,or both and can, under appropriate conditions, form a molecular complexcomprising the protein probe and the target protein. The region capableof binding to a target protein includes an antibody, a peptide, anaptamer, or a peptoid. The antibody can be obtained from a variety ofsources, including but not limited to polyclonal antibody, monoclonalantibody, monospecific antibody, recombinantly expressed antibody,humanized antibody, plantibodies, and the like. The terms protein,polypeptide, peptide, and amino acid sequence are used interchangeablyherein to refer to polymers of amino acids of any length. The polymermay be linear or branched, it may comprise modified amino acids, and itmay be interrupted by non-amino acids or synthetic amino acids. Theterms also encompass an amino acid polymer that has been modified, forexample, by disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term aminoacid refers to either natural and/or unnatural or synthetic amino acids,including but not limited to glycine and both the D or L opticalisomers, and amino acid analogs and peptidomimetics. Probes and methodsfor binding and identifying a target protein have been described, e.g.,in US2011/0086774, the contents of which is incorporated herein byreference in its entirety.

In embodiments, a probe is prepared by a cysteine bioconjugation methodthat is stable, site-specific to, preferably, the antibody'shinge-region heavy-chain. This preparation method provides relativelycontrollable labeled oligonucleotides to antibody stoichiometric ratios.A probe can comprise a plurality (i.e., more than one, e.g., 2, 3, 4, 5,or more) labeled oligonucleotides per antibody. Generally, “heavier”probes, which comprise 3 or 4 labeled oligonucleotides per antibody, aresignificantly less sensitive than antibodies lacking a labeledoligonucleotide or “lighter” probes, which comprise 1 or 2 labeledoligonucleotides per antibody.

Protein-targeting probes and nucleic acid-targeting probes may beapplied simultaneously as long as conditions allow for binding of both aprotein target and a nucleic acid target. Alternately, protein-targetingprobes and nucleic acid-targeting probes may be applied sequentiallywhen conditions allowing for binding of both a protein target and anucleic acid target are not possible.

A set of probes is synonymous with a composition of probes. A set ofprobes includes at least one species of probes, i.e., directed to onetarget. A set of probes preferably includes at least two, e.g., 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or morespecies of probes. A probe set may include one or multiple copies ofeach species of probe.

A first set of probes only may be applied to a sample. Alternately, asecond set (or higher number) of probes may be later applied to thesample. The first set and second (or higher number) may target onlynucleic acids, only proteins, or a combination thereof.

In the present invention, two or more targets (i.e., proteins, nucleicacids, or a combination thereof) are detected; 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 or more targets, and anynumber there between, are detected.

A set of probes may be pre-defined based upon the cell type or tissuetype to be targeted. For example, if the tissue is a breast cancer, thenthe set of probes will include probes directed to proteins relevant tobreast cancer cells (e.g., Her2, EGFR, and PR) and/or probes directed toproteins relevant to normal breast tissues. Additionally, the set ofprobes may be pre-defined based upon developmental status of a cell ortissue to be targeted. Alternately, the set of probes may be pre-definedbased upon subcellular localizations of interest, e.g., nucleus,cytoplasm, and membrane. For example, antibodies directed to Foxp3,Histone H3, or P-S6 label the nucleus, antibodies directed to CD3, CD4,PD-1, or CD4SRO label the cytoplasm, and antibodies directed to PD-L1label membranes.

A probe may be chemically synthesized or may be produced biologicallyusing a vector into which a nucleic acid encoding the probe has beencloned.

Any probe or set of probes described herein may be used in methods andkits of the present invention.

For the herein-described probes, association of label code to targetnucleic acid or target protein is not fixed.

Probes of the present invention can be used to detect a target nucleicacid or protein present in any sample, e.g., a biological sample. Aswill be appreciated by those in the art, the sample may comprise anynumber of things, including, but not limited to: cells (including bothprimary cells and cultured cell lines) and tissues (including culturedor explanted). In embodiments, a tissue sample (fixed or unfixed) isembedded, serially sectioned, and immobilized onto a microscope slide.As is well known, a pair of serial sections will include at least onecell that is present in both serial sections. Structures and cell types,located on a first serial section will have a similar location on anadjacent serial section. The sample can be cultured cells or dissociatedcells (fixed or unfixed) that have been immobilized onto a slide.

In embodiments, a tissue sample is a biopsied tumor or a portionthereof, i.e., a clinically-relevant tissue sample. For example, thetumor may be from a breast cancer. The sample may be an excised lymphnode.

The sample can be obtained from virtually any organism includingmulticellular organisms, e.g., of the plant, fungus, and animalkingdoms; preferably, the sample is obtained from an animal, e.g., amammal. Human samples are particularly preferred.

In some embodiments, the probes, compositions, methods, and kitsdescribed herein are used in the diagnosis of a condition. As usedherein the term diagnose or diagnosis of a condition includes predictingor diagnosing the condition, determining predisposition to thecondition, monitoring treatment of the condition, diagnosing atherapeutic response of the disease, and prognosis of the condition,condition progression, and response to particular treatment of thecondition. For example, a tissue sample can be assayed according to anyof the probes, methods, or kits described herein to determine thepresence and/or quantity of markers of a disease or malignant cell typein the sample (relative to the non-diseased condition), therebydiagnosing or staging a disease or a cancer.

In general, samples attached to a slide can be first imaged usingfluorescence (e.g., fluorescent antibodies or fluorescent stains (e.g.,DAPI)) to identify morphology, regions of interest, cell types ofinterest, and single cells and then expression of proteins and/ornucleic acids can be digitally counted from the sample on the sameslide.

Compositions and kits of the present invention can include probes andother reagents, for example, buffers and other reagents known in the artto facilitate binding of a protein and/or a nucleic acid in a sample,i.e., for performing hybridization reactions.

A kit also will include instructions for using the components of thekit, including, but not limited to, information necessary to hybridizelabeled oligonucleotides to a probe, to hybridize a probe to atarget-specific oligonucleotide, to hybridize a target-specificoligonucleotide to a target nucleic acid and/or to hybridize a probe totarget protein.

An exemplary protocol for detecting target nucleic acids and/or targetproteins is described as follows and as shown in FIGS. 10 to 14 (top).

Cells (live or fixed) or tissue sections (e.g., formalin-fixed paraffinembedded (FFPE)) that are prepared consistent with multiplexedimmunohistochemistry methods and/or nucleic acid in situ hybridizationmethods are prepared and immobilize onto a glass slide or suitable solidsupport. Access to the surface of cells or tissue-section is preserved,allowing for fluidic exchange; this can be achieved by using a fluidicchamber reagent exchange system (e.g., Grace™ Bio-Labs, Bend Oreg.).Regions-of-interest (ROIs) are identified on the serial section to beprovided probes or on an adjacent serial section. In the first instance,full “macroscopic-features” imaging methodology to cell/tissues ofinterest is performed, e.g., DAPI staining, membrane staining,mitochondrial staining, specific epitope staining, and specifictranscript staining, to determine overall macroscopic features ofcell/tissue of interest. Alternately, regions-of-interest (ROIs) areidentified on a serial section adjacent to the serial section to beprovided the probes; here, full “macroscopic-features” imaging (asdescribed above) is performed on a first serial section (section #1 inFIGS. 11 and 12). This imaging will generally identifyregions-of-interest on the adjacent serial section (red line in panel Bin FIG. 10 and green oval and green triangle of section #2 in FIGS. 11and 12) where signal oligonucleotides will be released from the probesupon application of a suitable and directed force. Serial sections maybe approximately 5 μm to 15 μm from each other.

FIG. 13 and FIG. 14 (top) further illustrate steps of the presentinvention. Steps shown in FIG. 13 include the following. (1) Process:FFPE slide mounted tissue is incubated with a cocktail of primaryantibodies conjugated to DNA oligos via a photo-cleavable linker,together with a limited number of visible-wavelength imaging reagents.(2) View: Regions of interest (ROI) are identified with visible-lightbased imaging reagents at low-plex to establish overall “architecture”of tumor slice (e.g., image nuclei and/or using one or two key tumorbiomarkers). (3) Profile: Select ROIs are chosen for high-resolutionmultiplex profiling and oligos from the selected region are releasedfollowing exposure to UV light. (4) Plating: Free photocleaved oligosare then collected, e.g., via a microcapillary-based “sipper”, andstored in a microplate well for subsequent quantitation. (5) DigitallyCount: During the digital counting step, photocleaved oligos from thespatially resolved ROIs in the microplate are hybridized to 4-color,6-spot optical barcodes, enabling up to ˜1 million digital counts of theprotein targets (distributed over up to 800-plex markers) in a singleROI using standard NanoString nCounter® read-out instrument (e.g.,SPRINT, Flex, and MAX).

A region of interest may be a tissue type present in a sample, a celltype, a cell, or a subcellular structure within a cell.

A composition comprising a set of probes, each probe comprising areleasable signal oligonucleotide, is applied to the serial section. Theset of probes or may include probes that target proteins, target nucleicacids, or both. The composition may include capture probes. When probesindirectly bind to a target (protein and/or nucleic acid), the appliedcomposition includes intermediary oligonucleotides. The composition willinclude other reagents known in the art to facilitate binding of aprotein and/or a nucleic acid in a sample.

Blocking steps are performed before and/or after the composition isapplied.

For probes including photo-cleavable linkers, the solid support (e.g.,microscope slide) is placed in a microscope that is capable of providingexcitation light at a wavelength capable of cleaving the photo-cleavablelinker. A first region-of-interest (red line in panel B in FIG. 10 andROI_(i) in FIGS. 11 and 12) is excited with the light, thereby cleavingthe photo-cleavable linker and releasing the signal oligonucleotides. Asillustrated in FIGS. 6 and 9, a signal oligonucleotide includes at leasta region of a probe that presently has positions bound with at least onelabeled oligonucleotide or the nucleic acid from a probe that is boundor can be bound by a reporter probe. By directing excitation light onlyto ROIi, signal oligonucleotides are only released from probes withinROIi and not from probes located outside of ROIi, which retain theirsignal oligonucleotides. Thus, signal oligonucleotides are collectedonly for probes that are bound to targets within ROIi, therebypermitting detection of the identities and quantities of the targets(proteins and/or nucleic acids) located within ROIi.

The surface of the section is washed with small amount of buffer (˜5 to30 μl) and the eluate (containing the released signal oligonucleotides)is collected into a first sample container (shown as Sample “i” in FIG.12). The surface of the section is further rinsed to remove any releasedsignal oligonucleotides that were omitted from the eluate.

A second region-of-interest (ROI_(j) in FIGS. 11 and 12) is excited withlight, thereby cleaving the photo-cleavable linker and releasing thesignal oligonucleotides from the second region-of-interest. Again, bydirecting excitation light only to ROIj, signal oligonucleotides areonly released from probes within ROIj and not from probes locatedoutside of ROIj, which retain their signal oligonucleotides. Thus,signal oligonucleotides are collected only for probes that are bound totargets within ROIj, thereby permitting detection of the identities andquantities of the targets (proteins and/or nucleic acids) located withinROIj.

The surface of the section is washed with small amount of buffer (˜5 to30 μl) and the eluate (containing the released signal oligonucleotides)is collected into a first sample tube (shown as Sample “j” in FIG. 12).The surface of the section is further rinsed to remove any releasedsignal oligonucleotides that were omitted from the eluate.

The excitation step, washing step, and rinsing step are repeated untilsignal oligonucleotides from all regions-of-interest (up to ROI_(n))have been collected.

Additional advantages, features, and embodiments of the presentinvention are illustrated in the Appendix filed herewith. As examples,various methods and devices for collecting a signal oligonucleotide andvarious ways of providing a force are shown. Moreover, the Appendixprovides unexpectedly improved results obtained from certain embodimentsof the present invention over other embodiments. Data demonstratingabout 7-fold to about 200-fold signal-to-noise improvements are shown.

Detection can use any microscope-type device or system known in the art.A device or system may include wide field illumination along with adigital mirror device (DMD; see FIGS. 15 and 16); advantages of thisinclude reduced costs since the DMD and controller can also drive theLED (which photocleaves probes) and adds essentially no additional cost,provides ease of implementation, allows small feature size of ˜1 mmwhich will include 10-40 mm cells, and leverages available consumerelectronics (like projectors). A device or system may include a laserscanning device, e.g., confocal, see FIG. 16. An advantage of this issmaller morphological features can be illuminated and imaged; however,additional costs are involved with these devices.

The plurality of target proteins and/or target nucleic acids present ineach region of interest in a sample are identified in each eluate sampleusing a polymerase reaction, a reverse transcriptase reaction,hybridization to an oligonucleotide microarray, mass spectrometry,hybridization to a fluorescent molecular beacon, a sequencing reaction,or nCounter® Molecular Barcodes. nCounter® systems and methods fromNanoString Technologies®, as described in US2003/0013091,US2007/0166708, US2010/0015607, US2010/0261026, US2010/0262374,US2010/0112710, US2010/0047924, US2014/0371088, and US2011/0086774), area preferred means for identifying target proteins and/or target nucleicacids. nCounter® systems and methods from NanoString Technologies® allowsimultaneous multiplexed identification a plurality (800 or more)distinct target proteins and/or target nucleic acids.

Together, a comparison of the identity and abundance of the targetproteins and/or target nucleic acids present in first region of interest(e.g., tissue type, a cell type (including normal and abnormal cells),and a subcellular structure within a cell) and the identity andabundance of the target proteins and/or target nucleic acids present insecond region of interest or more regions of interest can be made.

The present invention provides multiplexed detection and comparison ofup to 800 proteins of interest from discrete regions within a tumor (forexample) and its adjacent normal tissue; thus, enabling systematicinterrogation of the tumor and its microenvironment.

The present invention can be used in ongoing clinical studies toelucidate novel responses to immunotherapies and other targetedtherapies.

The present invention also enables the discovery of immune biomarkers intumors (for example) which can be used in the development of companiondiagnostics.

Immunohistochemistry is a powerful technique for analyzing proteinexpression and localization in FFPE tissue sections. However, it suffersfrom a number of challenges, including a lack of dynamic range,difficult quantitation, and labor intensive workflow for very limitedmultiplexing. Here is disclosed a novel platform based on the nCounter®barcoding technology which enables spatially-resolved, digitalcharacterization of proteins in a highly multiplexed (up to 800-plex)assay, i.e., the nCounter® Digital Multiplexed Immunohistochemistry(IHC) assay. The assay relies upon antibodies coupled to photo-cleavableoligonucleotide tags which are released from discrete regions of atissue using focused through-objective UV (e.g., ˜365 nm) exposure.Cleaved tags are quantitated in an nCounter® assay and counts mappedback to tissue location, yielding a spatially-resolved digital profileof protein abundance. The protein-detection may be performed along withor separate from a nucleic acid-detection assay which uses nucleic acidprobes comprising photo-cleavable oligonucleotide tags. Thus, thepresent invention can provide spatially-resolved digital profile ofprotein abundance, spatially-resolved digital profile of protein andnucleic acid abundance, or spatially-resolved digital profile of nucleicacid abundance.

Advantages of the assay include, but are not limited to: highsensitivity (e.g., ˜1 to 4 cells), all digital counting, with largedynamic range (>10⁵), highly multiplexed (e.g., 30 targets and scalable,with no change in instrumentation, to 800 targets), simple workflow,compatibility with FFPE, no secondary antibodies (for protein detection)or amplification reagents, and potential for clinical assays.

As used in this Specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive and covers both “or” and “and”.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromthe context, all numerical values provided herein are modified by theterm “about.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although other probes,compositions, methods, and kits similar, or equivalent, to thosedescribed herein can be used in the practice of the present invention,the preferred materials and methods are described herein. It is to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

EXAMPLES Example 1: The Present Invention Provides a“Barcoding-Potential” to Quantify Multiplexed Targets in a FFPE TissueSection

Intratumoral heterogeneity has emerged as a critical challenge to theimplementation of targeted therapeutics. Historically,immunohistochemistry (IHC) has been used to assess spatial heterogeneityof proteins; however, it has been difficult to quantify proteinabundance at high multiplex and wide dynamic range.

In this example, proteins in a formalin-fixed paraffin embedded (FFPE)tissue section were labeled with antibody-comprising probes thatincluded photo-cleavable linkers and fluorescent barcodes. The probes—ina user-defined ROI of the FFPE tissue section—were subsequently exposedto focused UV light, thereby releasing the signal oligonucleotides(comprising the fluorescent barcodes) from the ROI. The released signaloligonucleotides were washed away from the FFPE sample and collected.The fluorescent barcodes from the released signal oligonucleotides werethen recognized and digitally counted by an nCounter® system fromNanoString Technologies®, thereby quantifying the abundance of eachtargeted protein in the user-defined spatial region of a tissue section.After the signal oligonucleotides from a first ROI were released andcollected, the focused UV light was exposed to a second user-defined ROIof the FFPE tissue section, thereby releasing the signaloligonucleotides from the second ROI. In this non-limiting example, ahigh degree of linearity (0.97<R²<0.99) for the number of observedcounts versus area of UV illumination was observed and with a detectionspatial resolution of about 100 μm×100 μm, or approximately 100 cells.Unexpectedly, the present invention provides a “barcoding-potential” toquantify up to 800 targets with 5.5 logarithms (base 10) of dynamicrange in a single FFPE tissue section.

Example 2: The Present Invention Provides a Practical and FeasibleApproach for Quantifying Protein Expression Without Signal Amplificationand for Achieving Higher-Order Target Antigen Multiplexing in a FFPETissue Section

Quantitative, multiplexed immunohistochemistry has emerged as an area ofgreat interest within oncology since it has the unique capability ofidentifying spatiotemporal organization and interdependencies thatfurther define how checkpoint blockade impacts tumor microenvironment.This example describes a one-step, amplification-free staining methodusing a photo-cleavable oligo-tagged primary antibody which interactswith the target antigen within an FFPE tissue section. Illumination withultraviolet (UV) light is applied which releases the oligo from theantibody and is followed by eluent collection, quantification, anddigital counting that corresponds to antigen abundance.

First was investigated a variety of conjugation methods; thisestablished a cysteine bioconjugation method that is stable,site-specific to predominantly the hinge-region heavy-chain, andrelatively controllable in terms of oligonucleotide to antibodystoichiometric ratios.

Next was performed a linear regression analysis to determine therelationship between UV-induced cleavage area and measured digitalprotein counts; from this was observed a high degree of linearity(0.97<R²<0.99), confirming the basic mechanism/premise associated withthis multiplexed protein counting method on FFPE tissue.

To determine the impact of the presence of a conjugated oligonucleotideon antibody-antigen interaction, the performance of a labeledoligonucleotides-conjugated antibody to the unmodified antibody underidentical conditions in FFPE tissue sections was compared in terms ofsensitivity, specificity and signal intensity. Antibodies were selectedthat targeted antigens localized to the nucleus, cytoplasm, or membraneto determine the relationship between antibody performance andsubcellular location of target antigens. Selected antibodies targetedFoxp3, Histone H3, P-S6 (nuclear antigens), CD3, CD4, PD-1, CD45RO(cytoplasmic antigens), and PD-L1 (membranous antigen). In terms ofsensitivity, generally, “heavier” oligonucleotide-conjugated antibodies(having 3 or 4 labeled oligonucleotide per antibody) were found to besignificantly less sensitive when compared to unconjugated antibodies or“lighter” oligonucleotide-conjugated antibodies (having 1 or 2 labeledoligonucleotide per antibody). No significant difference was observedbetween unconjugated or “lighter” oligonucleotide-conjugated antibodiesin terms of sensitivity, specificity, or intensity across nuclear,cytoplasmic and membranous target antigens.

The present invention provides highly multiplexed protein profiling thatmeasures absolute protein expression levels using practical and feasiblemethods to comprehensively define the immune landscape in tumors beforeand during immunotherapeutic intervention.

Example 3: The Present Invention Provides Spatially-Resolved,Multiplexed Protein Detection from FFPE Tissue

Methods

Antibodies—Antibodies used in this Example and Examples 4 to 6 mayinclude: “target (clone ID, vendor))”: H3 (D1H2, CST), CD8 (OTI3H6,Origene), CD4 (SP35, Spring Bio), FOXP3 (D2W8E, CST), B7-H3 (D9M2L,CST), S6 (54D2, CST), B7-H4 (D1M8I, CST), Granzyme B (OTI4E4, Origene),Ki67 (8D5, CST), PD-1 (Nat105, Cell Marque), CD3 (MRQ-39, Cell Marque),Vista (D1L2G, CST), Her2 (29D8, CST), PR (D8Q2J, CST), ER (SP1, SpringBio), EGFR (D38B1, CST), CD56 (MRQ-42, Cell Marque), PD-L1 (E1L3N, CST),CD45 (2B11&PD7/26, Cell Marque), TIM-3 (D5D5R, CST), and Pan Keratin(C11, CST), CD45RO (UCHL1, Cell Marque).

Tonsil Microscopy—5 μm sections of a tonsil FFPE block (Amsbio) weremounted on slides. IHC was performed using standard protocols. Antigenretrieval was performed with a pressure cooker. Staining of the tonsilsection was performed with CD3 primary antibody MRQ-39 (Rabbit mAb, CellMarque) and Ki-67 primary antibody 8D5 (Mouse mAb, CST). Secondaryincubations were performed with Alexa594 labeled Goat α Rabbit (LifeTech.) and Alexa488 labeled Goat α Mouse (Life Tech.)

Here, samples attached to a slide were first imaged using fluorescentantibodies and then expression of proteins was digitally counted fromthe sample.

Steps similar to those illustrated in FIG. 10 to FIG. 14 (top) wereused. UV-cleavage of selected ROIs allowed full 30-plex digitalprofiling (nCounter® counts).

Results

FIG. 18 shows a photomicrograph establishing overall tissue morphologyof a tonsil sample that was initially imaged using 2-color fluorescenceof Ki-67 (cell proliferation marker; in green) and CD3 (immune cellmarker; in red). Multiple targets analyzed across twelve regions(including the four regions magnified in FIG. 19) show three distinctprofiles of Ki-67 and CD3 localization. FIG. 19 shows nCounter® countsfor Ki-67 and CD3 for four regions shown in FIG. 18. FIG. 20 showsexemplary counts from a 30-plex oligo-antibody cocktail on the twelveregions of interest (ROI) from the tonsil sample shown in FIG. 18. Datawas obtained from serial sections (to allow various additional controlsto be examined). As shown, regions of the tissue sample can beclassified based on the intensity and identity of the markers expressed.Exemplary classifications shown: “CD3-enriched”, “Ki67-enriched”,“Mixed”, and “Connective tissue”.

These data show that the present invention provides spatially-resolveddetection of a plurality (here, at least 30) of protein markers. Byscaling up the number of protein probes (antibodies) used, up to 800different protein markers can be detected and with similar resolution.

Example 4: The Present Invention Provides Multiplexed Protein Detectionfrom FFPE Tissue and Approaching Single-Cell Resolution

Methods

Melanoma Microscopy—5 μm sections of a melanoma (lymph node derived)FFPE block (Asterand) were mounted on slides. IHC was performed usingstandard protocols. Antigen retrieval was performed with a pressurecooker.

Here, samples were first imaged using fluorescence and then expressionof proteins was digitally counted from the sample.

Steps similar to those illustrated in FIG. 10 to FIG. 14 (top) wereused.

Results

FIG. 21 shows a photomicrograph establishing overall tissue morphologyof T cells in a melanoma sample of lymph node that was initially imagedusing three-color fluorescence of CD3 (in red), CD8 (in green), and DAPI(in blue). The white circle is 25 82 m in diameter and surrounds threecells.

FIG. 22 shows nCounter® data of CD3 conjugate release from FFPE lymphnode tissue section (5 μm thickness) as a function of UV illuminationarea (100 μm to 1 mm in diameter). The limit of detection counts(LOD=background counts+2× standard deviation) corresponds to spatialresolution of 26 μm in diameter. The field diaphragm size is shown belowthe figure. FIG. 23 and FIG. 24 show data for CD45 and PD1(respectively, from the same experiment).

The data shows a spatial detection ability of the present inventioncorresponding to about one to four cells.

Example 5: The Present Invention Provides Quantitative Performance in aClinically-Relevant Assay

Method

Steps similar to those illustrated in FIG. 10 to FIG. 14 (top) wereused.

Breast Cancer tissue microarray (TMA): TMA BR1504a obtained from USBiomax, Inc., H&E staining image obtained from US Biomax website (WorldWide Web (www) biomax.ushissue-arrays/Breast/BR1504a). Section from thesame block as the section shown on in the left panel of FIG. 25 werestained with Her2 primary antibody 29D8 (Rabbit mAb, CST), and Alexa594labeled Goat a Rabbit (Life Tech.). Counts were also obtained forHistone H3, Ribsomal Protein S6, Estrogen Receptor, ProgesteroneReceptor, Mouse IgG isotype control, and Rabbit IgG isotype control(data not shown). Her2 pathologist scores for TMA BR1504a were providedby US Biomax, Inc. (World Wide Web (www)biomax.us/tissue-arrays/Breast/BR1504a). Staining was performed withHer2 primary antibody 29D8 (Rabbit mAb, CST), and Alexa594 labeled Goata Rabbit (Life Tech.). Although other Rabbit primary antibodies wereused in the primary cocktail, fluorescence from these antibodies wasnegligible compared to Her2 fluorescence. Sum Pixel Intensities (atλ=594) were obtained using ImageJ software. For this, the backgroundvalue was set to intensity=0 and the highest intensity was set tointensity=255. The summation of all pixel intensities per ROI is shown.

Here, samples were first imaged using fluorescence and then expressionof proteins was digitally counted from the sample.

Results

FIG. 25 (left panel) shows a tissue microarray (TMA) of breast tumortissue containing variable levels of Her2 protein as shown in thephotomicrograph (center panel) which identifies Her2 fluorescence by IHCstaining. The right panel shows a magnification of a single region ofthe central panel; such regions were stained with a multiplexed antibodycocktail.

FIG. 26 shows nCounter® count data for forty-eight representativeregions versus Her2 status (ASCO-CAP guidelines). FIG. 27 plotsnCounter® Counts versus Sum Pixel Intensities (x10³) for the forty-eightregions mentioned above.

These digital count data show a high correlation with fluorescenceintensities (R2=0.92, FIG. 27) compared to visual Her2 status scoringvia ASCO-CAP guidelines (R2=0.51, FIG. 26).

Example 6: The Present Invention Reveals Abundances of Specific CellTypes in a Tissue Sample

Steps similar to those illustrated in FIG. 10 to FIG. 14 (top) wereused; a melanoma sample attached to a slide was first imaged usingfluorescence and then expression of proteins was digitally counted fromthe sample.

FIG. 28 shows a photomicrograph establishing overall tissue morphologyof a melanoma sample using two-color fluorescence of CD3 (immune cellmarker; in red) and DAPI (cell nuclei, in blue). Expression data using a30 antibody cocktail was obtained from the ten regions identified withwhite boxes. FIG. 29 shows exemplary nCounter® counts from a 30 -plexoligo-antibody cocktail on the ten regions of interest (ROI) from themelanoma sample shown in FIG. 28. Counts for thirteen markers, eachhaving expression counts above background, are shown. Regions 5, 6, 7,identified as “Immune infiltrate-enriched” have the highest expressionof T-cell markers and T-cell regulatory markers.

These data show that the present invention provides spatially-resolveddetection of a plurality (here, at least 30) of protein markers. Byscaling up the number of protein probes (antibodies) used, up to 800different protein markers can be detected and with similar resolution.

Example 7: A Digital Mirror Device (DMD) is Capable of IlluminatingSingle Cells

FIGS. 30 and 31 are photomicrographs showing that UV illumination usinga digital mirror device (DMD) is capable if illuminating single cells ina tonsil tissue sample.

These data show that the present invention is capable of single cellresolution when using a DMD.

Example 8: A Gel Box is Capable of Illuminating an Entire Sample andReleasing Signal Oligonucleotides From Probes Bound to the Entire Sample

FIG. 34: Shows an embodiment in which a whole tissue or sample isilluminated, e.g., with a standard laboratory UV gel box. Here, a FFPEtissue slide was placed on the light panel, a wax pen was used to holdbuffer solution (TBS) covering the FFPE tissue, and UV light exposure(276-362 nm, e.g., 302 nm; ˜5 mW/cm²) was applied to the tissue throughthe glass slide (1 mm thickness). The data shows that within about oneminute of UV exposure, most of signal oligonucleotides are released fromFFPE bound antibodies. Counts are normalized to a positive control.

Example 9: Illumination From a Microscope is Capable of Illuminating aRegion of Interest in a Sample and Releasing Signal OligonucleotidesFrom Probes Bound to the Region of Interest

FIG. 35 shows an embodiment in which a portion of a tissue or sample isilluminated, e.g., with a microscope, i.e., UV cleavage under Microscope(Time titration experiment). This is in contrast to the experiment ofExample 8, in which a whole sample is illuminated. Here, UV LED (at 365nm) is applied at about ˜150 mW/cm² with a 20× objective. UVillumination scans the whole tissue area identified by previousfluorescence (˜590 nm excitation) bright field imaging. Within about onesecond of UV exposure per field of view (FOV), most signaloligonucleotides are released from FFPE bound probes. The gel boxexperiment of Example 8 was utilized as a non-spatially resolved 100%release control. Counts are normalized ratio to positive control. Blue:microscope data with variable lengths of exposure time; Red: Gel box 2.5minutes exposure data. Also shown are photographs and a schematicshowing configuration of the microscope apparatus.

FIG. 36 shows signal oligonucleotides are released from a uniformlydistributed anti-Histone(H3) antibody bound to lung tissue sample.Tissue was exposed to one second of UV (365 nm, ˜150 mW/cm with 20×objective) per field-of-view (FOV) of about 450 μm×330 μm=0.15 mm².“Macro-Volume” used to collect effluent was about 70 μl. This decreasesthe limit of detection to (FOV/5)˜99 μm×99 μm with collection effluentof about 5 μl. Hence, in this example, the limit of detection isapproximately 10 cells×10 cells niche. These data show that antibodysignal is proportional to spatially resolved illumination area FOV andestimate “macro-fluidics” limit-of-detection (LOD).

FIG. 37 shows an embodiment in which a portion of a tissue or sample isilluminated, e.g., with a microscope, i.e., UV cleavage under microscope(Illumination area titration experiment) and for multiple targets. Shownis UV cleavage of multiple targets in a tissue: two positive targets(Histone H3 and Ribosomal S6) and eight 8-negative targets. Only onenegative target (0×40), showed high background. Data from zero, one,four, nine and sixteen fields of view are shown.

Example 10: A Region of Interest May be Pre-Identified by a LabelingTechnique and Then the Region of Interest is Illuminated SignalOligonucleotides are Released from Probes Bound to the Pre-IdentifiedRegion of Interest

FIG. 38 shows an embodiment in which a region of interest in a tissue(e.g., a breast cancer sample) is first identified for expression of amarker (here Her2) and this region of interest is then illuminated(e.g., with UV) to release signal oligonucleotides from a bound probe.Data shown compares the amount of signal oligonucleotides, for twotargets (here, Her2 and Histone H3), released from two locations: oneregion of interest that was pre-identified as Her2+ and one that waspre-identified as Her2−.

Example 11: A Sample Embedded in a Flow Cell Provides Collection ofElution From the Entire Sample and Not Only From a Region of InterestThat is Illuminated and From Which Signal Oligonucleotides are Released

FIG. 39 shows an embodiment in which a tissue is embedded in flow cell.Here, FFPE Tissue embedded in microfluidic flow cell (a 9 mm circularchamber with volume of 100 μm height with an approximate 25 μl volume[when the flow cell has a 300 μm height the approximate volume is 75μl]) controlled by a syringe pump. UV cleavage inside flow cell, showingelution profile illumination one area (9 FOVs) and elution, thenillumination another area (9 FOVs) and elution. Data for multiplefractions is shown. As with the data of Example 10, here a region ofinterest was pre-identified for expression of a fluorescently-labeledmarker.

Example 12: A Sample Embedded in a Flow Cell Comprising Small Holes Overa Region of Interest Provides Efficient Collection of Elution From theRegion of Interest That is Illuminated and Where Signal OligonucleotidesAre Released and Not From the Entire Sample

FIG. 40 shows an embodiment in which a tissue is embedded in flow cellwith small holes. Here, elution occurs directly above the region ofinterest. 0.4-1 mm diameter holes above fluidic chamber allow collectionof eluent (e.g., 5 μl collection volume). Tested were 9-hole, 96-holeformat, and 12-hole format (for tissue microarray (TMA)). Thefluorescence image was created by combining multiple fields of view.Also shown are photographs and a schematic showing configuration of theapparatus.

FIGS. 41A to 41C shows that embodiments using a flow cell with smallholes have significant signal to noise improvement rather thancollection of eluate from entire surface of tissue. The data shows thatcollecting eluent through a hole above a region of interest increasessignal-to-noise by about 7 fold. In this embodiment, 1 mm diameter holesabove fluidic flow cell (25 μl chamber) were used to collect eluent (5μl fractions). Data for multiple fractions is shown.

FIGS. 42A to 42C shows data in the using a flow cell with small holes(12 or 96 hole formats). The data shows that collecting eluent through ahole above a region of interest increases signal-to-noise by about 7fold. In this embodiment, field of view illumination was focused at thecenter of a hole; 5 μl volume of elution per hole.

FIGS. 43A and B shows data in comparing background signal from flowcells in which whole tissue elution was performed (FIG. 43A; as inExample 11) and background signal from flow cells in which elutionoccurred directly above a region of interest (FIG. 43B). As seen in FIG.43A, there is higher background for the whole tissue elution relative tothe background seen in the FIG. 43B. Additionally, FIG. 43B shows nodifference between in-flow cell and non-flow cell incubation.

Example 13: Released Signal Oligonucleotides can be Elected Via a SingleTube/Pipet, a Plurality of Tubes/Pipets, or a Multi-Tube/Pipet Array

FIG. 44 is a schematic showing eluent collection with an open surfacefor a multi-region of interest aspiration embodiment. Here is shown amulti-tube array for aspiration/dispensing eluents with rotary valveselection. See also, FIG. 47.

FIG. 45 includes photographs and a schematic showing an embodiment inwhich eluent collection is through a capillary (micro-aspirator). Seealso, FIG. 47. FIGS. 46A and B shows data from the embodiment of FIG. 45in which eluent collection is through a capillary (micro-aspirator).This embodiment has a dramatic improvement in signal to noise: signal tonoise ratio increases about 10 fold, compared to flow cell through holeelution and signal to noise ratio increases about 200 fold, compared towhole tissue elution. Here, the LOD area is approximately 60 μm×60 μm.

Example 14: A Device Comprising Both Illuminating and ElutionCapabilities can Efficiently and Accurately Obtain Nucleic Acid and/orProtein Expression Data From a Defined Region of Interest

FIG. 48 is a schematic showing illumination and fluid collection througha combined capillary and lens.

Example 15: Protein Expression Can Be Detected and Quantified From aSingle Cell

FIG. 50 shows protein expression data obtained from a single cell or twocells using the herein described methods and apparatuses. In the toppanel, S6 protein is detected and quantified from at least one cell andin the bottom panel, CD45 protein is detected and quantified from atleast one cell.

Example 16: The Herein Described Methods and Apparatuses Provide anAccurate and Efficient Detection and Quantification ofSpatially-Resolved, Multiplexed RNA Target and/or Protein TargetExpression

In situ hybridization (ISH) was performed to hybridize DNA oligo-basedprobes (“RNA probes”), each comprising a target-binding domain, a signaloligonucleotide, and a photo-cleavable linker, to an endogenous RNA. 5μm FFPE HER2 3+ breast tissue sections were deparaffinized in xylene,partially rehydrated in graded ethanols, and incubated in 70% ethanolfor 1 hour at room temperature. Then sections were incubated in 40μg/m1proteinase K for 25 minutes at 37° C. Tissues were then incubated in 50%formamide/2×SSC for 15 minutes at room temperature and hybridizedovernight at 37C in a solution of 1 nM probes, 40% formamide, 1 mg/mlyeast tRNA, 10% dextran sulfate, and 0.2% BSA in 2×SSC. Afterhybridization, two stringent washes in 50% formamide/2×SSC wereperformed for 25 minutes each at 37° C. Sections were stained withTO-PRO®-3 (Thermo Fisher Scientific) fluorescent nucleic acid stain tovisualize tissue morphology. Focused UV light, directed by a digitalmicromirror device, was then used to cleave DNA signal oligonucleotidesfrom probes in a user-defined region of interest (ROI). For each tissuesection, two ROIs comprised a tumorous tissue, two ROIs comprised normaltissue, and two ROIs comprised no tissue at all (histology slideitself). After cleavage, signal oligonucleotides were collected,hybridized to nCounter® Molecular Barcodes, and digitally counted by annCounter® system from NanoString Technologies®. H&E was performed ontissue sections to verify tumorous and normal tissue ROIs.

On serial sections, standard immunohistochemistry (IHC) was performedusing “Protein probes,” each comprising an antibody as target-bindingdomain, a DNA signal oligonucleotide, and a photo-cleavable linker.Sections were then stained with an anti-rabbit Alexa 594 secondaryantibody and TO-PRO®-3 (Thermo Fisher Scientific) fluorescent nucleicacid stain to visualize tissue morphology. Focused UV light, directed bya digital micromirror device (DMD), was then used to cleave DNA signaloligonucleotides from probes in a user-defined ROI. For each tissuesection, two ROIs comprised tumorous tissue, one ROI comprised normaltissue, and two ROIs comprised no tissue at all (histology slideitself). ROIs were matched to the ROIs selected for ISH probe cleavage.Following cleavage, the signal oligonucleotides from protein targetswere mixed with the signal oligonucleotides from RNA targets and allwere quantitated as described above. H&E was performed on tissuesections to verify tumorous and normal tissue ROIs and to verify ROIswere correctly matched between ISH and IHC tissues.

FIG. 51 shows ROIs sampled from serial sections of the same tumorsample. Regions 1-4 are not shown in this image and, instead, were takenfrom portions of the tissue that did not contain tissue (negativecontrols—“No Tissue”). Regions 5-8 contained low numbers of tumor cells(“Normal Tissue”). Regions 9-12 contained high numbers of tumor cells(“Tumor”).

FIG. 52 shows counts obtained for six of the nine RNA probes included inthis assay. For each ROI, a sample was collected prior to applying theUV illumination (the “−UV” set of data) and prior to collecting a plusUV sample from the same region (the “+UV” set of data). Backgroundlevels of counts are obtained when UV was not applied to the sample;thus showing the UV-dependence of an obtained signal. ROIs that were+UV, but not directed to tissue (i.e., ROIs 1-4—“No Tissue”) gavebackground counts. Regions that were primarily normal tissue (i.e., ROIs5-8—“Normal Tissue”) gave low counts for the HER2 probe (orange bars onthe graph). Regions that were primarily tumor tissue (i.e., ROIs9-12—“Tumor”) gave higher counts for HER2. A similar, but less dramatic,increase was seen for the Ribosome S6 probe (green bars in graph).Additional control probes targeted RNAs not expected to be expressedhighly in this tissue type gave consistent counts that did not showdifferential levels between Normal and Tumor Tissue. These controlprobes were designed to target CD45, PSA (Prostate-Specific Antigen),and two unique ERCC sequences. For clarity, FIG. 53 shows the averagesand standard deviations of data shown in FIG. 52.

These RNA probe samples were also run simultaneously with Protein probesthat analyzed the sample regions of the tumor sample. For this, RNA andProtein probes were simultaneously hybridized to nCounter® MolecularBarcodes, and digitally counted by an nCounter® system from NanoStringTechnologies®. Counts for this assay are shown in FIG. 54. An increasein HER2 RNA probe counts (Red bars in top graph) and Protein probecounts (Red and orange bars in bottom graph) are seen in the Tumorregions compare to the normal regions. Only +UV samples are shown. The−UV control sample, as described above, are not shown in this graphbecause they gave background counts (similar to “No Tissue” counts). ROI6 and ROI 8 were dropped from this analysis because matching Proteinprobe samples were not obtained. Thus, signal oligonucleotides fromProtein probes and signal oligonucleotides from RNA probes can bedetected and quantified together.

Example 17: Partially Double-Stranded Probes Have Higher Signal-To-NoiseRatios When Compared to Single-Stranded Probes

DNA probes (that recognize and bind to mRNA) were hybridized in situ, asdescribed in Example 16, to RNA in 5 μm FFPE tissues. UV cleavage wasperformed on whole tissue sections, mounted on separate slides, for 3minutes using a UV light box (gel box) in 2×SSC+0.1% Tween 20. Aftercleavage and release of the signal oligonucleotides, the signaloligonucleotides were collected by a pipette and detected as in Example16. Single-stranded DNA probes, partially double-stranded DNA probes,and no probe controls counts are shown for HER2 3+ breast tissue andtonsil tissue in FIG. 55 (top graph). Signal-to-noise ratios weredetermined by dividing counts by average background counts (average ERCCcounts); see, FIG. 55, bottom graph.

Example 18: Addition of Salmon Sperm DNA Improves Probe Hybridization

DNA probes (that recognize and bind to mRNA) were hybridized in situ, asdescribed above, to RNA in 5 μm FFPE tissues. 1 mg/ml sonicated,denatured salmon sperm DNA was used instead of yeast tRNA duringhybridization. Slides were hybridized with a solution of 1nM probes, 40%formamide, 1 mg/ml sonicated, denatured salmon sperm DNA, 10% dextransulfate, and 0.2% BSA in 2×SSC. UV cleavage and signal oligonucleotidecollection and detection were performed as described in Example 17.Single stranded DNA probes are shown in HER2 3+ breast and tonsil (FIG.56). Signal-to-noise ratios were determined by dividing counts byaverage background counts (average ERCC counts).

Example 19: PSA (Prostate-Specific Antigen) RNA Probe is Highly Specific

DNA probes (that recognize and bind to mRNA) were hybridized in situ, asdescribed above, to RNA in 5 μm sections of FFPE prostate. A ten minuteincubation in IVIES for at 97° C. was used instead of a one hour ethanolincubation. UV cleavage, signal oligonucleotide collection anddetection, and signal-to-noise ratio calculations were performed asdescribed in Example 17. Counts and ratios are shown in FIG. 57.

Example 20: Specificity of Probes Increase at Non-Standard, Sub-nMConcentrations

Typically, in situ hybridization (ISH) probes that are used to recognizeRNA are hybridized at 5 to 200 nM. Surprisingly, nucleic acidrecognizing-probes of the present invention performed best at, or below,0.2 nM, which is 25 to 1000-fold lower than standard ISH probeconcentrations.

DNA probes were hybridized to RNA in situ, as described above, in 5 μmsections of FFPE HER2 3+ breast samples. Probes were used at 5, 1, 0.2,and 0.4 nM. UV cleavage, signal oligonucleotide collection anddetection, and fold change calculation were performed as described inExample 17.

FIG. 58 shows that counts decreased with decreasing probe concentrations(top graph). However, unexpectedly, there was a significant gain insignal-to-noise when positive probe counts are compared to negativecontrol probes, when probes are hybridized at sub-nM concentrations.

What is claimed is:
 1. A method comprising: (1) contacting at least oneprotein target in or from at least one cell in a sample with at leastone probe comprising a target-binding domain and a signaloligonucleotide; (2) providing a force to a location of the samplesufficient to release the signal oligonucleotide; and (3) collecting andidentifying the released signal oligonucleotide, thereby detecting theat least one protein target in or from a specific location of the samplethat was provided the force.
 2. The method of claim 1, wherein detectingcomprises determining the identity and the abundance of the at least oneprotein target.
 3. The method of claim 2, wherein the at least oneprotein target comprises at least two distinct protein targets or atleast two copies of the same protein target.
 4. The method of claim 3,wherein detecting comprises comparing the abundance of each distinctprotein target.
 5. The method of claim 1, further comprising repeatingat least steps (2) and (3) on at least a second specific location of thesample, the second specific location comprising at least a second cell.6. The method of claim 5, wherein detecting comprises comparing theabundance of the at least one protein target in or from the specificlocation and in or from the at least second specific location.
 7. Themethod of claim 6, wherein the at least one cell and the at least secondcell are the same cell type.
 8. The method of claim 6, wherein the atleast one cell and the at least second cell are distinct cell types. 9.The method of claim 8, wherein detecting comprises comparing theabundance of the at least one protein target in or from a first celltype and in or from the at least second cell type.
 10. The method ofclaim 9, wherein the first cell type and the at least second cell typeare independently selected from a normal cell and an abnormal cell. 11.The method of any one of claims 1 to 10, wherein the at least one cellis directly immobilized to a surface or is indirectly immobilized to thesurface via at least one other cell.
 12. The method of claim 11, whereinthe sample is a 2 to 1000 μm thick tissue section.
 13. The method ofclaim 12, wherein the tissue section is obtained from a formalin-fixedparaffin embedded (FFPE) sample.
 14. The method of any one of claims 1to 11, wherein the at least one cell is a cultured cell, a primary cell,or a dissociated cell from an explant.
 15. The method of claim 12 or 14,wherein the at least one cell is fixed or unfixed.
 16. The method of anyone of claims 1 to 15, wherein the at least one cell is stained orlabeled prior to step (2) thereby allowing visualization of asubcellular, cellular, or tissue-related structure in the stained orlabeled cell.
 17. The method of any one of claims 1 to 16, wherein theat least one probe further comprises a linker located between thetarget-binding domain and the signal oligonucleotide.
 18. The method ofany one of claims 1 to 17, wherein the signal oligonucleotide is asingle-stranded nucleic acid or a partially double-stranded nucleicacid.
 19. The method of any one of claims 1 to 18, wherein a negativepurification is used to remove intact probe molecules from the releasedsignal oligonucleotides.
 20. The method of claim 19, wherein thenegative purification comprises an affinity purification comprisingcontacting an intact probe with an immobilized oligonucleotide that iscomplementary to a portion of the intact probe or an immobilizedantibody or protein-binding motif that recognizes and binds to a portionof the intact probe.
 21. The method of claim 20, wherein the intactprobe's target binding domain comprises a universal purification tag orsequence that is partially complementary to the immobilizedoligonucleotide or is capable of being recognized or bound by theimmobilized antibody or protein-binding motif.
 22. The method of any oneof claims 1 to 21, wherein the linker comprises a cleavable linker. 23.The method of claim 22, wherein the cleavable linker is photo-cleavable.24. The method of claim 23, wherein the provided force is light.
 25. Themethod of claim 24, wherein the light is provided by a light sourceselected from the group consisting of an arc-lamp, a laser, a focused UVlight source, and light emitting diode (LED).
 26. The method of claim25, wherein the light irradiates at least one subcellular structure ofthe at least one cell.
 27. The method of any one of claims 1 to 26,wherein detecting comprises determining the abundance of the at leastone protein target in or from the at least one subcellular structure ofthe at least one cell.
 28. The method of claim 5, wherein the force islight which is provided by a light source selected from an arc-lamp, alaser, a focused UV light source, and a light emitting diode (LED) andthe light source irradiates at least one subcellular structure in the atleast one cell and at least one subcellular structure in the at leastsecond cell.
 29. The method of claim 28, wherein detecting comprisescomparing the abundance of the at least one protein target in or fromthe at least one subcellular structure in the at least one cell and theat least one subcellular structure in the at least second cell.
 30. Themethod of any one of claims 1 to 29, wherein the target-binding domainis selected from the group consisting of an antibody, a peptide, anaptamer, and a peptoid.
 31. The method of any one of claims 1 to 30,wherein detecting comprises a polymerase reaction, a reversetranscriptase reaction, hybridization to an oligonucleotide microarray,mass spectrometry, hybridization to a fluorescent molecular beacon, asequencing reaction, or nCounter® Molecular Barcodes.
 32. The method ofany one of claims 1 to 31, wherein the protein target is an intactprotein, a plurality of polypeptides, a polypeptide, or a peptide. 33.The method of any one of claims 1 to 32, wherein the signaloligonucleotide is collected from the sample via liquid laminar,turbulent, or transitional flow in a channel of 25 to 500 μm depthbetween the sample and a fluidic device or impermeable barrier placedover the sample.
 34. The method of any one of claims 1 to 33, whereinthe released signal oligonucleotide is collected from a solutionproximal to the at least one cell.
 35. The method of claim 34, whereinthe proximal solution is at least immediately above the at least onecell.
 36. The method of claim 35, wherein the proximal solution iscollected by aspirating.
 37. The method of claim 36, wherein theaspirating is via a pipette, a capillary tube, a microarray pin, and/ora flow cell comprising holes.
 38. The method of claim 37, wherein thecapillary tube comprises an optical device capable of transmitting alight force to the at least one cell.
 39. The method of claim 38,wherein the light force is UV light.
 40. The method of claim 39, whereinthe pipette or microarray pin is attached to an array comprising aplurality of pipettes or microarray pins.
 41. The method of any one ofclaims 1 to 40, wherein the proximal solution comprises an anionicpolymer, preferably dextran sulfate, and/or salmon sperm DNA.
 42. Themethod of any one of claims 1 to 41, wherein the collected signaloligonucleotide is added to a solution comprising an anionic polymer,preferably dextran sulfate, and/or salmon sperm DNA.
 43. The method ofany one of claims 1 to 42, wherein the method further comprisesilluminating a region of interest using a laser scanning device.
 44. Themethod of any one of claims 1 to 42, wherein the method furthercomprises illuminating a region of interest using a digital mirrordevice (DMD).
 45. The method of any one of claims 1 to 44, wherein themethod provides simultaneous spatially resolved protein detection of asample.
 46. The method of any one of claims 1 to 45, wherein digitalreadout comprises a linear dynamic range of >5 logs.
 47. The method ofany one of claims 1 to 46, wherein the sample is attached to a slide andis first imaged using fluorescence and then expression of proteins isdigitally counted from the sample.
 48. The method of any one of claims 1to 47, wherein the probe is provided at a concentration of 5 nM or less.49. The method of claim 48, wherein the probe is provided at aconcentration of 1 nM or less.
 50. The method of claim 49, wherein theprobe is provided at a concentration of 0.4 nM or less.
 51. The methodof claim 50, wherein the probe is provided at a concentration of 0.2 nMor less.